Immunohistochemical Detection of the Autophagy Markers LC3 and p62/SQSTM1 in Formalin-Fixed and Paraffin-Embedded Tissue

  • Sabina BerezowskaEmail author
  • José A. Galván
Part of the Methods in Molecular Biology book series (MIMB, volume 1560)


Autophagy is a highly conserved cellular mechanism of “self digestion,” ensuring cellular homeostasis, and playing a role in many diseases including cancer. As a stress response mechanism, it may also be involved in cellular response to therapy.

LC3 and Sequestosome 1 (p62/SQSTM1) are among the most widely used markers to monitor autophagy, and can be visualized in formalin-fixed and paraffin-embedded tissue by immunohistochemistry. Here we describe a validated staining protocol using an automated staining system available in many routine pathology laboratories, enabling high-throughput staining under standardized conditions.

Key words

Immunohistochemistry LC3 Sequestosome 1 p62/SQSTM1 Autophagy 



This work was supported by a grant from the Bernese Cancer League to SB.


  1. 1.
    Mizushima N, Levine B, Cuervo AM et al (2008) Autophagy fights disease through cellular self-digestion. Nature 451(7182):1069–1075CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Scarlatti F, Granata R, Meijer AJ et al (2009) Does autophagy have a license to kill mammalian cells? Cell Death Differ 16(1):12–20CrossRefPubMedGoogle Scholar
  3. 3.
    Tschan MP, Simon HU (2010) The role of autophagy in anticancer therapy: promises and uncertainties. J Intern Med 268(5):410–418CrossRefPubMedGoogle Scholar
  4. 4.
    Feng Y, He D, Yao Z et al (2014) The machinery of macroautophagy. Cell Res 24(1):24–41CrossRefPubMedGoogle Scholar
  5. 5.
    Hurley JH, Schulman BA (2014) Atomistic autophagy: the structures of cellular self-digestion. Cell 157(2):300–311CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Klionsky DJ, Abdelmohsen K, Abe A et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kabeya Y, Mizushima N, Ueno T et al (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Schlafli AM, Berezowska S, Adams O et al (2015) Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry. Eur J Histochem 59(2):2481CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Martinet W, Schrijvers DM, Timmermans JP et al (2013) Immunohistochemical analysis of macroautophagy: recommendations and limitations. Autophagy 9(3):386–402CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Mijaljica D, Nazarko TY, Brumell JH et al (2012) Receptor protein complexes are in control of autophagy. Autophagy 8(11):1701–1705CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Komatsu M, Ichimura Y (2010) Physiological significance of selective degradation of p62 by autophagy. FEBS letters 584(7):1374–1378CrossRefPubMedGoogle Scholar
  12. 12.
    Zois CE, Giatromanolaki A, Sivridis E et al (2011) "Autophagic flux" in normal mouse tissues: focus on endogenous LC3A processing. Autophagy 7(11):1371–1378CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institute of PathologyUniversity of BernBernSwitzerland

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